Milk-based nutritional compositions containing lactoferrin and uses thereof

ABSTRACT

The present disclosure relates to milk-based nutritional compositions comprising lactoferrin and/or a prebiotic component, wherein, when combined, the lactoferrin and prebiotic component may exhibit additive or synergistic beneficial effects on the health and development of a pediatric subject. The disclosure further relates to methods comprising the administration of said milk-based nutritional compositions to pediatric subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 12/980,813, filed Dec. 29, 2010, the disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates generally to milk-based nutritionalcompositions comprising lactoferrin that are suitable for administrationto pediatric subjects. Additionally, the disclosure relates to methodsof supporting and promoting gastrointestinal health, immune function,cognitive development and brain function and to a method of reducingpsychological stress in a pediatric subject via administration of amilk-based nutritional composition comprising lactoferrin and aprebiotic component, wherein the lactoferrin and the prebiotic componentmay exhibit additive and/or synergistic beneficial effects.

BACKGROUND ART

Lactoferrin, an iron-binding glycoprotein, is one of the majormultifunctional agents present in human milk. It has the capacity tobind two molecules of iron in a reversible fashion and can facilitatethe uptake of iron within the intestines. Further, lactoferrin has beenshown to be both bacteriostatic and bactericidal, and it aids inpreventing intestinal infections in humans, especially in pediatricsubjects.

Moreover, human lactoferrin has been reported to protect againstGram-negative bacteria in a variety of ways. It is believed that humanlactoferrin exerts a bacteriostatic activity by depriving microorganismsof the iron that is necessary for growth. Thus, by sequestering theenvironmental iron of pathogenic microorganisms, human lactoferrineffectively inhibits the growth of those microorganisms.

Several studies have examined the effect of human lactoferrin on variousbacterial species. For example, a 2001 study demonstrated that humanlactoferrin can inhibit the adhesion of EPEC to HeLa cells. Nascimentode Arujao, A., et al., Lactoferrin and Free Secretory Component of HumanMilk Inhibit the Adhesion of Enteropathogenic Escherichia coli to HeLaCells, BMC Microbiol. 1:25 (2001).

Further, human lactoferrin appears to have a positive effect on thesymptoms of diarrheal diseases. Yet some women are unwilling or unableto breastfeed. Accordingly, in an effort to mimic breast milk, it wouldbe beneficial to provide a nutritional composition for a pediatricsubject that contains lactoferrin from a non-human source. However, theaddition of lactoferrin to commercially viable nutritional compositionshas generally been limited due to lactoferrin's proclivity to losefunctional capacity during formula processing steps that involvesignificant fluctuation in temperature and/or pH.

Moreover, the infant gut microflora is rapidly established in the firstfew weeks following birth, and it has a great impact on an infant'simmune system. The nature of this intestinal colonization is initiallydetermined by early exposure to environmental sources of microbes and bythe general state of health of the infant. Whether the infant isbreast-fed or formula-fed also has a strong influence on the intestinalbacterial population.

Human milk contains a number of factors that may contribute to thegrowth and population of the gut microflora of infants. Among thesefactors is a complex mixture of more than 130 different oligosaccharidesthat reach levels as high as 8-12 g/L in transitional and mature milk.Kunz, et al., Oligosaccharides in Human Milk Structure, Functional, andMetabolic Aspects, Ann. Rev. Nutr. 20: 699-722 (2000). Theseoligosaccharides are resistant to enzymatic digestion in the uppergastrointestinal tract and reach the colon intact, where they then serveas substrates for colonic fermentation.

Cow's milk and commercially available infant formulas that are based oncow's milk provide only trace amounts of oligosaccharides; as a result,prebiotics may be used to supplement the diet of formula-fed infants.Prebiotics have been defined as non-digestible food ingredients thatbeneficially affect the host by selectively stimulating the growthand/or activity of one or a limited number of cells in the colon thatcan improve the health of the host.

Both the interaction among dietary components and among the microfloraof the intestinal ecosystem are very complex. Consequently, then, thematrix of an infant formula or other pediatric nutritional compositionmay influence the effectiveness of prebiotics when such ingredients areprovided as supplements in the diet of a formula-fed infant. Further,the type and concentration of proteins used in a formula matrix may alsomodulate the intestinal microbiota. Because human milk is the preferredsource of infant nutrition, it is desirable to provide a formula matrixthat mimics the qualities of human milk by allowing for effectivesupplementation of prebiotics as functional food ingredients.

Accordingly, it would be beneficial to provide a nutritional compositionfor pediatric subjects that contains both lactoferrin and prebiotics.

BRIEF SUMMARY

Briefly, the present disclosure is directed, in an embodiment, to amethod for modulating psychological stress in a pediatric subject, themethod comprising administering to the pediatric subject a milk-basednutritional composition comprising lactoferrin from a non-human source.In certain embodiments, the method comprises administering:

a. up to about 7 g/100 kcal of a fat or lipid source, more preferablyabout 3 g/100 kcal to about 7 g/100 kcal of a fat or lipid source;

b. up to about 5 g/100 kcal of a protein source, more preferably about 1g/100 kcal to about 5 g/100 kcal of a protein source;

c. at least about 10 mg/100 kcal of lactoferrin, more preferably fromabout 70 mg/100 kcal to about 220 mg/100 kcal of lactoferrin, and mostpreferably about 90 mg/100 kcal to about 190 mg/100 kcal of lactoferrin;and

d. about 0.1 g/100 kcal to about 1 g/100 kcal of a prebiotic compositioncomprising polydextrose and/or galactooligosaccharide.

In certain embodiments, the disclosure is directed to a method formodulating plasma corticosterone levels in a pediatric subject, themethod comprising administering to the pediatric subject a milk-basednutritional composition comprising bovine lactoferrin.

In some embodiments, the disclosure is directed to a method forsupporting gastrointestinal development in a pediatric subject, themethod comprising administering to the pediatric subject a milk-basednutritional composition comprising bovine lactoferrin and a prebioticcomponent comprising polydextrose.

In still other embodiments, the disclosure is directed to methods forsupporting healthy growth and development in a pediatric subject byadministering to the subject a nutritional composition comprisinglactoferrin and at least one prebiotic.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the disclosureand are intended to provide an overview or framework for understandingthe nature and character of the disclosure as it is claimed. Thedescription serves to explain the principles and operations of theclaimed subject matter. Other and further features and advantages of thepresent disclosure will be readily apparent to those skilled in the artupon a reading of the following disclosure.

DETAILED DESCRIPTION

Reference now will be made in detail to the embodiments of the presentdisclosure, one or more examples of which are set forth hereinbelow.Each example is provided by way of explanation of the nutritionalcomposition of the present disclosure and is not a limitation. In fact,it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope of the disclosure. Forinstance, features illustrated or described as part of one embodiment,can be used with another embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent disclosure are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only and is not intended as limiting the broader aspects ofthe present disclosure.

The present disclosure relates generally to milk-based nutritionalcompositions comprising lactoferrin that are suitable for administrationto a pediatric subject. Additionally, the disclosure relates to methodsof supporting and promoting gastrointestinal health, immune function,cognitive development and brain function and to methods of reducingpsychological stress in a pediatric subject via administration ofmilk-based nutritional compositions comprising lactoferrin and aprebiotic component.

“Nutritional composition” means a substance or formulation thatsatisfies at least a portion of a subject's nutrient requirements. Theterms “nutritional(s)”, “nutritional formula (s)”, “enteralnutritional(s)”, and “nutritional supplement(s)” are used asnon-limiting examples of nutritional composition(s) throughout thepresent disclosure. Moreover, “nutritional composition(s)” may refer toliquids, powders, gels, pastes, solids, concentrates, suspensions, orready-to-use forms of enteral formulas, oral formulas, formulas forinfants, formulas for pediatric subjects, formulas for children,growing-up milks and/or formulas for adults.

The term “enteral” means deliverable through or within thegastrointestinal, or digestive, tract. “Enteral administration” includesoral feeding, intragastric feeding, transpyloric administration, or anyother administration into the digestive tract. “Administration” isbroader than “enteral administration” and includes parenteraladministration or any other route of administration by which a substanceis taken into a subject's body.

“Pediatric subject” means a human less than 13 years of age. In someembodiments, a pediatric subject refers to a human subject that isbetween birth and 8 years old. In other embodiments, a pediatric subjectrefers to a human subject between 1 and 6 years of age. In still furtherembodiments, a pediatric subject refers to a human subject between 6 and12 years of age. The term “pediatric subject” may refer to infants(preterm or full term) and/or children, as described below.

“Infant” means a human subject ranging in age from birth to not morethan one year and includes infants from 0 to 12 months corrected age.The phrase “corrected age” means an infant's chronological age minus theamount of time that the infant was born premature. Therefore, thecorrected age is the age of the infant if it had been carried to fullterm. The term infant includes low birth weight infants, very low birthweight infants, extremely low birth weight infants and preterm infants.“Preterm” means an infant born before the end of the 37th week ofgestation. “Late preterm” means an infant form between the 34th week andthe 36th week of gestation. “Full term” means an infant born after theend of the 37th week of gestation. “Low birth weight infant” means aninfant born weighing less than 2500 grams (approximately 5 lbs, 8ounces). “Very low birth weight infant” means an infant born weighingless than 1500 grams (approximately 3 lbs, 4 ounces). “Extremely lowbirth weight infant” means an infant born weighing less than 1000 grams(approximately 2 lbs, 3 ounces).

“Child” means a subject ranging in age from 12 months to about 13 years.In some embodiments, a child is a subject between the ages of 1 and 12years old. In other embodiments, the terms “children” or “child” referto subjects that are between one and about six years old, or betweenabout seven and about 12 years old. In other embodiments, the terms“children” or “child” refer to any range of ages between 12 months andabout 13 years.

“Children's nutritional product” refers to a composition that satisfiesat least a portion of the nutrient requirements of a child. A growing-upmilk is an example of a children's nutritional product.

The term “degree of hydrolysis” refers to the extent to which peptidebonds are broken by a hydrolysis method.

The term “partially hydrolyzed” means having a degree of hydrolysiswhich is greater than 0% but less than about 50%.

The term “extensively hydrolyzed” means having a degree of hydrolysiswhich is greater than or equal to about 50%.

The term “protein-free” means containing no measurable amount ofprotein, as measured by standard protein detection methods such assodium dodecyl(lauryl) sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) or size exclusion chromatography. In some embodiments, thenutritional composition is substantially free of protein, wherein“substantially free” is defined hereinbelow.

“Infant formula” means a composition that satisfies at least a portionof the nutrient requirements of an infant. In the United States, thecontent of an infant formula is dictated by the federal regulations setforth at 21 C.F.R. Sections 100, 106, and 107. These regulations definemacronutrient, vitamin, mineral, and other ingredient levels in aneffort to simulate the nutritional and other properties of human breastmilk.

The term “growing-up milk” refers to a broad category of nutritionalcompositions intended to be used as a part of a diverse diet in order tosupport the normal growth and development of a child between the ages ofabout 1 and about 6 years of age.

“Milk-based” means comprising at least one component that has been drawnor extracted from the mammary gland of a mammal. In some embodiments, amilk-based nutritional composition comprises components of milk that arederived from domesticated ungulates, ruminants or other mammals or anycombination thereof. Moreover, in some embodiments, milk-based meanscomprising bovine casein, whey, lactose, or any combination thereof.Further, “milk-based nutritional composition” may refer to anycomposition comprising any milk-derived or milk-based product known inthe art.

“Nutritionally complete” means a composition that may be used as thesole source of nutrition, which would supply essentially all of therequired daily amounts of vitamins, minerals, and/or trace elements incombination with proteins, carbohydrates, and lipids. Indeed,“nutritionally complete” describes a nutritional composition thatprovides adequate amounts of carbohydrates, lipids, essential fattyacids, proteins, essential amino acids, conditionally essential aminoacids, vitamins, minerals and energy required to support normal growthand development of a subject.

Therefore, a nutritional composition that is “nutritionally complete”for a preterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thepreterm infant.

A nutritional composition that is “nutritionally complete” for a fullterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of all carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thefull term infant.

A nutritional composition that is “nutritionally complete” for a childwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of a child.

As applied to nutrients, the term “essential” refers to any nutrientthat cannot be synthesized by the body in amounts sufficient for normalgrowth and to maintain health and that, therefore, must be supplied bythe diet. The term “conditionally essential” as applied to nutrientsmeans that the nutrient must be supplied by the diet under conditionswhen adequate amounts of the precursor compound is unavailable to thebody for endogenous synthesis to occur.

“Probiotic” means a microorganism with low or no pathogenicity thatexerts a beneficial effect on the health of the host.

The term “inactivated probiotic” means a probiotic wherein the metabolicactivity or reproductive ability of the referenced probiotic has beenreduced or destroyed. The “inactivated probiotic” does, however, stillretain, at the cellular level, at least a portion its biologicalglycol-protein and DNA/RNA structure. As used herein, the term“inactivated” is synonymous with “non-viable”.

“Prebiotic” means a non-digestible food ingredient that beneficiallyaffects the host by selectively stimulating the growth and/or activityof one or a limited number of bacteria in the digestive tract that canimprove the health of the host.

“Phytonutrient” means a chemical compound that occurs naturally inplants. Phytonutrients may be included in any plant-derived substance orextract. The term “phytonutrient(s)” encompasses several broadcategories of compounds produced by plants, such as, for example,polyphenolic compounds, anthocyanins, proanthocyanidins, andflavan-3-ols (i.e. catechins, epicatechins), and may be derived from,for example, fruit, seed or tea extracts. Further, the termphytonutrient includes all carotenoids, phytosterols, thiols, and otherplant-derived compounds. Moreover, as a skilled artisan will understand,plant extracts may include phytonutrients, such as polyphenols, inaddition to protein, fiber or other plant-derived components. Thus, forexample, apple or grape seed extract(s) may include beneficialphytonutrient components, such as polyphenols, in addition to otherplant-derived substances.

“β-glucan” means all β-glucan, including specific types of β-glucan,such as β-1,3-glucan or β-1,3;1,6-glucan. Moreover, β-1,3;1,6-glucan isa type of β-1,3-glucan. Therefore, the term “β-1,3-glucan” includesβ-1,3;1,6-glucan.

“Pectin” means any naturally-occurring oligosaccharide or polysaccharidethat comprises galacturonic acid that may be found in the cell wall of aplant. Different varieties and grades of pectin having varied physicaland chemical properties are known in the art. Indeed, the structure ofpectin can vary significantly between plants, between tissues, and evenwithin a single cell wall. Generally, pectin is made up of negativelycharged acidic sugars (galacturonic acid), and some of the acidic groupsare in the form of a methyl ester group. The degree of esterification ofpectin is a measure of the percentage of the carboxyl groups attached tothe galactopyranosyluronic acid units that are esterified with methanol.

Pectin having a degree of esterification of less than 50% (i.e., lessthan 50% of the carboxyl groups are methylated to form methyl estergroups) are classified as low-ester, low methoxyl, or low methylated(“LM”) pectins, while those having a degree of esterification of 50% orgreater (i.e., more than 50% of the carboxyl groups are methylated) areclassified as high-ester, high methoxyl or high methylated (“HM”)pectins. Very low (“VL”) pectins, a subset of low methylated pectins,have a degree of esterification that is less than approximately 15%.

“Pathogen” means an organism that causes a disease state or pathologicalsyndrome. Examples of pathogens may include bacteria, viruses,parasites, fungi, microbes or combination(s) thereof.

“Modulate” or “modulating” means exerting a modifying, controllingand/or regulating influence. In some embodiments, the term “modulating”means exhibiting an increasing or stimulatory effect on the level/amountof a particular component. In other embodiments, “modulating” meansexhibiting a decreasing or inhibitory effect on the level/amount of aparticular component.

All percentages, parts and ratios as used herein are by weight of thetotal formulation, unless otherwise specified.

All amounts specified as administered “per day” may be delivered in oneunit dose, in a single serving or in two or more doses or servingsadministered over the course of a 24 hour period.

The nutritional composition of the present disclosure may besubstantially free of any optional or selected ingredients describedherein, provided that the remaining nutritional composition stillcontains all of the required ingredients or features described herein.In this context, and unless otherwise specified, the term “substantiallyfree” means that the selected composition may contain less than afunctional amount of the optional ingredient, typically less than 0.1%by weight, and also, including zero percent by weight of such optionalor selected ingredient.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods and compositions of the present disclosure, includingcomponents thereof, can comprise, consist of, or consist essentially ofthe essential elements and limitations of the embodiments describedherein, as well as any additional or optional ingredients, components orlimitations described herein or otherwise useful in nutritionalcompositions.

As used herein, the term “about” should be construed to refer to both ofthe numbers specified as the endpoint(s) of any range. Any reference toa range should be considered as providing support for any subset withinthat range.

The present disclosure is directed to milk-based nutritionalcompositions comprising lactoferrin and/or a prebiotic component, touses thereof, and to methods comprising administration of thosenutritional compositions. The nutritional compositions of the presentdisclosure support overall health and development in a pediatric humansubject, such as an infant (preterm and/or term) or a child.

The intestinal microflora plays a crucial role in the postnataldevelopment of most gastrointestinal functions and also in thedevelopment of the gut-associated immune system in pediatric subjects.Accordingly, the nutritional composition of the present disclosuresupports gastrointestinal health and development. Moreover, a healthyintestinal flora supports an adequate gut-brain communication, affectingbrain function and, consequently, psychological stress responses,resulting in modified behavior. Administration of a prebiotic componentand lactoferrin together can modulate the intestinal flora of apediatric subject by increasing beneficial bacteria and/or reducingadhesion of pathogens in the gastrointestinal system. Thus, in someembodiments, the present disclosure is directed to a method formodulating psychological stress responses via administration of at leastone prebiotic component and lactoferrin. And in certain embodiments, thecombination of prebiotic(s) and lactoferrin has additive and/orsynergistic beneficial effects that support gastrointestinaldevelopment. In certain embodiments, the disclosure is related to amethod for supporting gastrointestinal health and/or development in apediatric subject. The method comprises administering lactoferrin and atleast one prebiotic to a pediatric subject.

In some embodiments, the present disclosure is directed to a method ofincreasing fecal secretory IgA in a pediatric subject by administeringto the subject a nutritional composition comprising a prebioticcomponent and lactoferrin. In certain embodiments, the presentdisclosure is directed to a method for modulating gastric emptyingand/or improving intestinal tolerance of enteral feeding in a pediatricsubject, the method(s) comprising administering a nutritionalcomposition comprising a combination of a prebiotic component andlactoferrin. In still other embodiments, the present disclosure isdirected to a method for reducing stool viscosity in a pediatric subjectby administering an effective amount of a prebiotic component togetherwith lactoferrin.

Additionally, the present disclosure is directed to a method forreducing the occurrence of necrotizing enterocolitis (NEC) in apediatric subject, the method comprising administering a prebioticcomponent and lactoferrin to a pediatric subject. In some embodiments,the reduced occurrence of NEC is associated with improvement information of an intestinal mucus layer in an infant via administrationof lactoferrin. More specifically, the inventors of the presentapplication have discovered that ileal production of mucin(s) may beincreased in an infant when a nutritional composition comprisinglactoferrin is administered to said infant. Thus, in some embodiments,the present disclosure is directed to a method for improving mucus layerformation in an infant, the method comprising administering an effectiveamount of lactoferrin to the infant. In other embodiments, the presentdisclosure is directed to a method for increasing ileal production ofmucin in a pediatric subject by administering a nutritional compositioncomprising lactoferrin and at least one prebiotic to the pediatricsubject.

Moreover, during early life, infants and children experience manystressful situations due to, for example, a changing environment orbeing hungry or tired. Consequently, stress hormones are released, whichmay negatively affect brain development and/or cause other long-termdetrimental effects in a pediatric subject. Yet the inventors of thepresent disclosure have discovered that administration of lactoferrin toa pediatric subject can reduce or suppress psychological stress and/ormodulate plasma corticosterone levels, thereby promoting healthy brainand cognitive growth and development in a pediatric subject.

Thus, in some embodiments, the present disclosure includes a method forreducing psychological stress in a pediatric subject comprisingadministering to the subject an effective amount of lactoferrin. Inother embodiments, the disclosure includes a method for modulatingplasma corticosterone levels in a pediatric subject, comprisingadministering to the pediatric subject an effective amount oflactoferrin. In still other embodiments, the disclosure is directed to amethod for improving brain and/or cognitive function in a pediatricsubject by administering an effective amount of lactoferrin to thesubject. In certain embodiments, the lactoferrin is administeredtogether with a prebiotic component that has additive and/or synergisticbeneficial effects when combined with the lactoferrin.

In some embodiments, brain development, brain functionality and,therefore, behavior may be modulated in a subject via administration oflactoferrin. Additionally, administration of lactoferrin may affect thehypothalamic-pituitary adrenal axis (HPA), its communication with thegastrointestinal system, the brain and/or other body systems. Ininfants, immaturity of the gut often causes gastrointestinal symptomsthat affect their nutritional status with consequences for their overallhealth. The present disclosure is directed to a nutritional compositionthat provides, in some embodiments, bovine lactoferrin and at least oneprobiotic, wherein administration of said nutritional compositionmodifies gut development and communication of the gut with the brain viathe gut-brain axis. Moreover, the nutritional compositions of thepresent disclosure may positively impact brain-related functions. Thus,in some embodiments, the present disclosure is directed to a method ofmodulating gut-brain communication, the method comprising administeringto a pediatric subject a nutritional composition comprising lactoferrin.

In certain other embodiments, the nutritional composition comprises acombination of Lactobacillus helveticus and Bifidobacterium longum.Administration of a nutritional composition comprising these probioticsto a pediatric subject may result in reduced anxiety in the subjectand/or in decreased serum cortisol in the subject, thereby indicatingbeneficial psychological effects. Accordingly, the present disclosure isdirected, in some embodiments, to a method for reducing anxiety in asubject, wherein the method comprises at least the step of administeringa nutritional composition comprising at least one probiotic selectedfrom the group consisting of Lactobacillus helveticus andBifidobacterium longum to a subject. In some embodiments, the presentdisclosure is directed to a method for decreasing serum cortisol in asubject, the method comprising administering a nutritional compositioncomprising lactoferrin and at least one probiotic selected from thegroup consisting of Lactobacillus helveticus and Bifidobacterium longumto the subject.

Further, in some embodiments, the nutritional composition may compriseLactobacillus rhamnosus. Administration of Lactobacillus rhamnosus to apediatric subject may reduce stress-induced corticosterone levels,thereby reducing the occurrence of anxiety and/or of depression-relatedbehaviors. As such, in some embodiments, the present disclosure isdirected to a method of reducing stress-induced corticosterone levels ina subject via administration of a nutritional composition comprisingLactobacillus rhamnosus to the subject.

In other embodiments, administration of lactoferrin modulates theintestinal barrier function in a subject. Moreover, in some embodiments,administration of lactoferrin modulates the immune system. In furtherembodiments, administration of lactoferrin modulates the opioid system.And in particular embodiments, lactoferrin behaves as an opioidantagonist. In still other embodiments, lactoferrin modulates otherphysiological processes, including satiety, food intake, and regulationof glucose and/or fat metabolism.

Furthermore, infants, particularly preterm infants, are often faced withsevere infections, such as sepsis, pneumonia and urinary tractinfection(s). Accordingly, as a result of the beneficial additive and/orsynergistic effects of administration of a prebiotic component togetherwith lactoferrin, preterm morbidity may be reduced. Therefore, in someembodiments, the present disclosure is directed to a method of reducingmorbidity in a preterm infant, comprising administering a nutritionalcomposition comprising lactoferrin to a preterm infant.

As used herein, “lactoferrin from a non-human source” means lactoferrinwhich is produced by or obtained from a source other than human breastmilk. For example, lactoferrin for use in the present disclosureincludes human lactoferrin produced by a genetically modified organismas well as non-human lactoferrin. The term “organism”, as used herein,refers to any contiguous living system, such as animal, plant, fungus ormicro-organism. The term “non-human lactoferrin”, as used herein, refersto lactoferrin having an amino acid sequence that is different than theamino acid sequence of human lactoferrin.

Lactoferrins are single chain polypeptides of about 80 kD containing 1-4glycans, depending on the species. The 3-D structures of lactoferrin ofdifferent species are very similar, but not identical. Each lactoferrincomprises two homologous lobes, called the N- and C-lobes, referring tothe N-terminal and C-terminal part of the molecule, respectively. Eachlobe further consists of two sub-lobes or domains, which form a cleftwhere the ferric ion (Fe³⁺) is tightly bound in synergistic cooperationwith a (bi)carbonate anion. These domains are called N1, N2, C1 and C2,respectively. The N-terminus of lactoferrin has strong cationic peptideregions that are responsible for a number of important bindingcharacteristics. Lactoferrin has a very high isoelectric point (˜pI 9)and its cationic nature plays a major role in its ability to defendagainst bacterial, viral, and fungal pathogens. There are severalclusters of cationic amino acids residues within the N-terminal regionof lactoferrin mediating the biological activities of lactoferrinagainst a wide range of microorganisms. For instance, the N-terminalresidues 1-47 of human lactoferrin (1-48 of bovine lactoferrin) arecritical to the iron-independent biological activities of lactoferrin.In human lactoferrin, residues 2 to 5 (RRRR) and 28 to 31 (RKVR) arearginine-rich cationic domains in the N-terminus especially critical tothe antimicrobial activities of lactoferrin. A similar region in theN-terminus is found in bovine lactoferrin (residues 17 to 42;FKCRRWQWRMKKLGAPSITCVRRAFA).

As described in “Perspectives on Interactions Between Lactoferrin andBacteria” which appeared in the publication BIOCHEMISTRY AND CELLBIOLOGY, pp 275-281 (2006), lactoferrins from different host species mayvary in their amino acid sequences though commonly possess a relativelyhigh isoelectric point with positively charged amino acids at the endterminal region of the internal lobe. Suitable lactoferrins for use inthe present disclosure include those having at least 48% homology withthe amino acid sequence AVGEQELRKCNQWSGL at the HLf (349-364) fragment.In some embodiments, the lactoferrin has at least 65% homology with theamino acid sequence AVGEQELRKCNQWSGL at the HLf (349-364) fragment, and,in embodiments, at least 75% homology. For example, non-humanlactoferrins acceptable for use in the present disclosure include,without limitation, bovine lactoferrin, porcine lactoferrin, equinelactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrinand camel lactoferrin.

Lactoferrin for use in the present disclosure may be, for example,isolated from the milk of a non-human animal or produced by agenetically modified organism. For example, in U.S. Pat. No. 4,791,193,incorporated by reference herein in its entirety, Okonogi et al.discloses a process for producing bovine lactoferrin in high purity.Generally, the process as disclosed includes three steps. Raw milkmaterial is first contacted with a weakly acidic cationic exchanger toabsorb lactoferrin followed by the second step where washing takes placeto remove nonabsorbed substances. A desorbing step follows wherelactoferrin is removed to produce purified bovine lactoferrin. Othermethods may include steps as described in U.S. Pat. Nos. 7,368,141,5,849,885, 5,919,913 and 5,861,491, the disclosures of which are allincorporated by reference in their entirety.

In one embodiment, lactoferrin is present in the nutritional compositionin an amount of at least about 10 mg/100 kCal. In certain embodiments,the nutritional composition may include between about 10 and about 240mg lactoferrin per 100 kCal. In another embodiment, where thenutritional composition is an infant formula, the nutritionalcomposition may comprise lactoferrin in an amount of from about 70 mg toabout 220 mg lactoferrin per 100 kCal; in yet another embodiment, thenutritional composition may comprise about 90 mg to about 190 mglactoferrin per 100 kCal. In still other embodiments, the nutritionalcomposition may comprise about 5 mg to about 16 mg lactoferrin per 100kcal. In further embodiments, the nutritional composition comprisesabout 9 mg to about 14 mg lactoferrin per 100 kcal.

In some embodiments, the nutritional composition can include lactoferrinin the quantities of from about 0.5 mg to about 1.5 mg per milliliter offormula. In nutritional compositions replacing human milk, lactoferrinmay be present in quantities of from about 0.6 mg to about 1.3 mg permilliliter of formula. In certain embodiments, the nutritionalcomposition may comprise between about 0.1 and about 2 grams lactoferrinper liter. In some embodiments, the nutritional composition includesbetween about 0.5 and about 1.5 grams lactoferrin per liter of formula.

The nutritional compositions described herein can, in some embodimentscomprise non-human lactoferrin, non-human lactoferrin produced by agenetically modified organism and/or human lactoferrin produced by agenetically modified organism. Lactoferrin is generally described as an80 kilodalton glycoprotein having a structure of two nearly identicallobes, both of which include iron binding sites. As described in“Perspectives on Interactions Between Lactoferrin and Bacteria” whichappeared in the publication BIOCHEMISTRY AND CELL BIOLOGY, pp 275-281(2006), lactoferrin from different host species may vary in an aminoacid sequence, though it commonly possesses a relatively highisoelectric point with positively charged amino acids at the endterminal region of the internal lobe. Lactoferrin has been recognized ashaving bactericidal and antimicrobial activities.

Surprisingly, the forms of lactoferrin included herein maintain relevantactivity even if exposed to a low pH (i.e., below about 7, and even aslow as about 4.6 or lower) and/or high temperatures (i.e., above about65° C., and as high as about 120° C., conditions which would be expectedto destroy or severely limit the stability or activity of humanlactoferrin or recombinant human lactoferrin. These low pH and/or hightemperature conditions can be expected during certain processing regimenfor nutritional compositions of the types described herein, such aspasteurization.

In some embodiments, the nutritional composition of the presentdisclosure comprises bovine lactoferrin. Bovine lactoferrin (bLF) is aglycoprotein that belongs to the iron transporter or transferringfamily. It is isolated from bovine milk, wherein it is found as acomponent of whey. There are known differences between the amino acidsequence, glycosylation patters and iron-binding capacity in human andbovine lactoferrin. Additionally, there are multiple and sequentialprocessing steps involved in the isolation of bovine lactoferrin fromcow's milk that affect the physiochemical properties of the resultingbovine lactoferrin preparation. Human and bovine lactoferrin are alsoreported to have differences in their abilities to bind the lactoferrinreceptor found in the human intestine.

In certain embodiments, the bLF has been isolated from whole milk havinga low somatic cell count. In some embodiments, “low somatic cell count”refers to a concentration of less than 200,000 cells/mL.

Though not wishing to be bound by this or any other theory, it isbelieve that bLF that has been isolated from whole milk has lesslipopolysaccharide (LPS) initially bound than does bLF that has beenisolated from milk powder. Additionally, it is believed that bLF with alow somatic cell count has less initially-bound LPS. A bLF with lessinitially-bound LPS has more binding sites available on its surface.This is thought to aid bLF in binding to the appropriate location anddisrupting the infection process.

The bLF that is used in certain embodiments may be any bLF isolated fromwhole milk and/or having a low somatic cell count, wherein “low somaticcell count” refers to a somatic cell count less than 200,000 cells/mL.By way of example, suitable bLF is available from Tatua Co-operativeDairy Co. Ltd., in Morrinsville, New Zealand, from FrieslandCampina Domoin Amersfoort, Netherlands or from Fonterra Co-Operative Group Limitedin Auckland, New Zealand.

In an embodiment, the bLF may be administered via a solution, capsule,tablet or caplet. Carriers for bLF can have a bLF concentration ofbetween about 0.01% and about 100%.

The nutritional composition may also contain one or more prebiotics(also referred to as a prebiotic component) in certain embodiments.Prebiotics exert health benefits, which may include, but are not limitedto, selective stimulation of the growth and/or activity of one or alimited number of beneficial gut bacteria, stimulation of the growthand/or activity of ingested probiotic microorganisms, selectivereduction in gut pathogens, and favorable influence on gut short chainfatty acid profile. Such prebiotics may be naturally-occurring,synthetic, or developed through the genetic manipulation of organismsand/or plants, whether such new source is now known or developed later.Prebiotics useful in the present disclosure may includeoligosaccharides, polysaccharides, and other prebiotics that containfructose, xylose, soya, galactose, glucose and mannose.

More specifically, prebiotics useful in the present disclosure mayinclude polydextrose, polydextrose powder, lactulose, lactosucrose,raffinose, gluco-oligosaccharide, inulin, fructo-oligosaccharide,isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,xylo-oligosaccharide, chito-oligosaccharide, manno-oligosaccharide,aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide,galacto-oligosaccharide, and gentio-oligosaccharides.

In an embodiment, the total amount of prebiotics present in thenutritional composition may be from about 1.0 g/L to about 10.0 g/L ofthe composition. More preferably, the total amount of prebiotics presentin the nutritional composition may be from about 2.0 g/L and about 8.0g/L of the composition. In some embodiments, the total amount ofprebiotics present in the nutritional composition may be from about 0.1g/100 kcal to about 1 g/100 kcal. In certain embodiments, the totalamount of prebiotics present in the nutritional composition may be fromabout 0.3 g/100 kcal to about 0.7 g/100 kcal. Moreover, the nutritionalcomposition may comprise a prebiotic component comprising polydextrose(“PDX”) In some embodiments, the prebiotic component comprises at least20% w/w PDX or a mixture thereof.

If PDX is used in the prebiotic composition, the amount of PDX in thenutritional composition may, in an embodiment, be within the range offrom about 0.1 g/100 kcal to about 1 g/100 kcal. In another embodiment,the amount of polydextrose is within the range of from about 0.2 g/100kcal to about 0.6 g/100 kcal. In some embodiments, PDX may be includedin the nutritional composition in an amount sufficient to providebetween about 1.0 g/L and 10.0 g/L. In another embodiment, thenutritional composition contains an amount of PDX that is between about2.0 g/L and 8.0 g/L. And in still other embodiments, the amount of PDXin the nutritional composition may be from about 0.1 mg/100 kcal toabout 0.5 mg/100 kcal or about 0.3 mg/100 kcal.

In other embodiments, the prebiotic component may comprisegalacto-oligosaccharide (GOS). If GOS is used in the prebioticcomposition, the amount of GOS in the nutritional composition may, in anembodiment, be from about 0.1 g/100 kcal to about 1 g/100 kcal. Inanother embodiment, the amount of GOS in the nutritional composition maybe from about 0.2 g/100 kcal to about 0.5 g/100 kcal. In otherembodiments, the amount of GOS in the nutritional composition may befrom about 0.1 mg/100 kcal to about 1.0 mg/100 kcal or from about 0.1mg/100 kcal to about 0.5 mg/100 kcal.

In a particular embodiment of the present invention, PDX is administeredin combination with GOS.

In a particular embodiment, GOS and PDX are supplemented into thenutritional composition in a total amount of at least about 0.2 mg/100kcal or about 0.2 mg/100 kcal to about 1.5 mg/100 kcal. In someembodiments, the nutritional composition may comprise GOS and PDX in atotal amount of from about 0.6 to about 0.8 mg/100 kcal.

Moreover, the nutritional composition(s) of the disclosure may compriseat least one protein source. The protein source can be any used in theart, e.g., nonfat milk, whey protein, casein, soy protein, hydrolyzedprotein, amino acids, and the like. Bovine milk protein sources usefulin practicing the present disclosure include, but are not limited to,milk protein powders, milk protein concentrates, milk protein isolates,nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, wheyprotein isolates, whey protein concentrates, sweet whey, acid whey,casein, acid casein, caseinate (e.g. sodium caseinate, sodium calciumcaseinate, calcium caseinate) and any combinations thereof.

In some embodiments, the proteins of the nutritional composition areprovided as intact proteins. In other embodiments, the proteins areprovided as a combination of both intact proteins and hydrolyzedproteins. In certain embodiments, the proteins are may be partiallyhydrolyzed or extensively hydrolyzed. In still other embodiments, theprotein source comprises amino acids. In yet another embodiment, theprotein source may be supplemented with glutamine-containing peptides.In another embodiment, the protein component comprises extensivelyhydrolyzed protein. In still another embodiment, the protein componentof the nutritional composition consists essentially of extensivelyhydrolyzed protein in order to minimize the occurrence of food allergy.In yet another embodiment, the protein source may be supplemented withglutamine-containing peptides.

Some people exhibit allergies or sensitivities to intact proteins, i.e.whole proteins, such as those in intact cow's milk protein or intact soyprotein isolate-based formulas. Many of these people with proteinallergies or sensitivities are able to tolerate hydrolyzed protein.Hydrolysate formulas (also referred to as semi-elemental formulas)contain protein that has been hydrolyzed or broken down into shortpeptide fragments and amino acids and as a result is more easilydigested. In people with protein sensitivities or allergies, immunesystem associated allergies or sensitivities often result in cutaneous,respiratory or gastrointestinal symptoms such as vomiting and diarrhea.People who exhibit reactions to intact protein formulas often will notreact to hydrolyzed protein formulas because their immune system doesnot recognize the hydrolyzed protein as the intact protein that causestheir symptoms.

Some gliadins and bovine caseins may share epitopes recognized byanti-gliadin IgA antibodies. Accordingly, then, the nutritionalcomposition of the present disclosure reduces the incidence of foodallergy, such as, for example, protein allergies and, consequently, theimmune reaction of some patients to proteins such as bovine casein, byproviding a protein component comprising hydrolyzed proteins, such ashydrolyzed whey protein and/or hydrolyzed casein protein. A hydrolyzedprotein component contains fewer allergenic epitopes than an intactprotein component.

Accordingly, in some embodiments, the protein component of thenutritional composition comprises either partially or extensivelyhydrolyzed protein, such as protein from cow's milk. The hydrolyzedproteins may be treated with enzymes to break down some or most of theproteins that cause adverse symptoms with the goal of reducing allergicreactions, intolerance, and sensitization. Moreover, the proteins may behydrolyzed by any method known in the art.

The terms “protein hydrolysates” or “hydrolyzed protein” are usedinterchangeably herein and refer to hydrolyzed proteins, wherein thedegree of hydrolysis is may be from about 20% to about 80%, or fromabout 30% to about 80%, or even from about 40% to about 60%. The degreeof hydrolysis is the extent to which peptide bonds are broken by ahydrolysis method. The degree of protein hydrolysis for purposes ofcharacterizing the hydrolyzed protein component of the nutritionalcomposition is easily determined by one of ordinary skill in theformulation arts by quantifying the amino nitrogen to total nitrogenratio (AN/TN) of the protein component of the selected formulation. Theamino nitrogen component is quantified by USP titration methods fordetermining amino nitrogen content, while the total nitrogen componentis determined by the Tecator Kjeldahl method, all of which are wellknown methods to one of ordinary skill in the analytical chemistry art.

When a peptide bond in a protein is broken by enzymatic hydrolysis, oneamino group is released for each peptide bond broken, causing anincrease in amino nitrogen. It should be noted that even non-hydrolyzedprotein would contain some exposed amino groups. Hydrolyzed proteinswill also have a different molecular weight distribution than thenon-hydrolyzed proteins from which they were formed. The functional andnutritional properties of hydrolyzed proteins can be affected by thedifferent size peptides. A molecular weight profile is usually given bylisting the percent by weight of particular ranges of molecular weight(in Daltons) fractions (e.g., 2,000 to 5,000 Daltons, greater than 5,000Daltons).

As previously mentioned, persons who exhibit sensitivity to whole orintact proteins can benefit from consumption of nutritional formulascontaining hydrolyzed proteins. Such sensitive persons may especiallybenefit from the consumption of a hypoallergenic formula.

In some embodiments, the nutritional composition of the presentdisclosure is substantially free of intact proteins. In this context,the term “substantially free” means that the preferred embodimentsherein comprise sufficiently low concentrations of intact protein tothus render the formula hypoallergenic. The extent to which anutritional composition in accordance with the disclosure issubstantially free of intact proteins, and therefore hypoallergenic, isdetermined by the August 2000 Policy Statement of the American Academyof Pediatrics in which a hypoallergenic formula is defined as one whichin appropriate clinical studies demonstrates that it does not provokereactions in 90% of infants or children with confirmed cow's milkallergy with 95% confidence when given in prospective randomized,double-blind, placebo-controlled trials.

Another alternative for pediatric subjects, such as infants, that havefood allergy and/or milk protein allergies is a protein-free nutritionalcomposition based upon amino acids. Amino acids are the basic structuralbuilding units of protein. Breaking the proteins down to their basicchemical structure by completely pre-digesting the proteins makes aminoacid-based formulas the most hypoallergenic formulas available.

In a particular embodiment, the nutritional composition is protein-freeand contains free amino acids as a protein equivalent source. In thisembodiment, the amino acids may comprise, but are not limited to,histidine, isoleucine, leucine, lysine, methionine, cysteine,phenylalanine, tyrosine, threonine, tryptophan, valine, alanine,arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine,proline, serine, carnitine, taurine and mixtures thereof. In someembodiments, the amino acids may be branched chain amino acids. In otherembodiments, small amino acid peptides may be included as the proteincomponent of the nutritional composition. Such small amino acid peptidesmay be naturally occurring or synthesized. The amount of free aminoacids in the nutritional composition may vary from about 1 to about 5g/100 kcal. In an embodiment, 100% of the free amino acids have amolecular weight of less than 500 Daltons. In this embodiment, thenutritional formulation may be hypoallergenic.

In a particular embodiment of the nutritional composition, thewhey:casein ratio of the protein source is similar to that found inhuman breast milk. In an embodiment, the protein source comprises fromabout 40% to about 85% whey protein and from about 15% to about 60%casein.

In some embodiments, the nutritional composition comprises between about1 g and about 7 g of a protein source per 100 kcal. In otherembodiments, the nutritional composition comprises between about 3.5 gand about 4.5 g of protein per 100 kcal.

Moreover, the nutritional composition of the present disclosure maycomprise at least one starch or starch component. A starch is acarbohydrate composed of two distinct polymer fractions: amylose andamylopectin. Amylose is the linear fraction consisting of α-1,4 linkedglucose units. Amylopectin has the same structure as amylose, but someof the glucose units are combined in an α-1,6 linkage, giving rise to abranched structure. Starches generally contain 17-24% amylose and from76-83% amylopectin. Yet special genetic varieties of plants have beendeveloped that produce starch with unusual amylose to amylopectinratios. Some plants produce starch that is free of amylose. Thesemutants produce starch granules in the endosperm and pollen that stainred with iodine and that contain nearly 100% amylopectin. Predominantamong such amylopectin producing plants are waxy corn, waxy sorghum andwaxy rice starch.

The performance of starches under conditions of heat, shear and acid maybe modified or improved by chemical modifications. Modifications areusually attained by introduction of substituent chemical groups. Forexample, viscosity at high temperatures or high shear can be increasedor stabilized by cross-linking with di- or polyfunctional reagents, suchas phosphorus oxychloride.

In some instances, the nutritional compositions of the presentdisclosure comprise at least one starch that is gelatinized orpregelatinized. As is known in the art, gelatinization occurs whenpolymer molecules interact over a portion of their length to form anetwork that entraps solvent and/or solute molecules. Moreover, gelsform when pectin molecules lose some water of hydration owing tocompetitive hydration of cosolute molecules. Factors that influence theoccurrence of gelation include pH, concentration of cosolutes,concentration and type of cations, temperature and pectin concentration.Notably, LM pectin will gel only in the presence of divalent cations,such as calcium ions. And among LM pectins, those with the lowest degreeof esterification have the highest gelling temperatures and the greatestneed for divalent cations for crossbridging.

Meanwhile, pregelatinization of starch is a process of precooking starchto produce material that hydrates and swells in cold water. Theprecooked starch is then dried, for example by drum drying or spraydrying. Moreover the starch of the present disclosure can be chemicallymodified to further extend the range of its finished properties. Thenutritional compositions of the present disclosure may comprise at leastone pregelatinized starch.

Native starch granules are insoluble in water, but, when heated inwater, native starch granules begin to swell when sufficient heat energyis present to overcome the bonding forces of the starch molecules. Withcontinued heating, the granule swells to many times its original volume.The friction between these swollen granules is the major factor thatcontributes to starch paste viscosity.

The nutritional composition of the present disclosure may comprisenative or modified starches, such as, for example, waxy corn starch,waxy rice starch, corn starch, rice starch, potato starch, tapiocastarch, wheat starch or any mixture thereof. Generally, common cornstarch comprises about 25% amylose, while waxy corn starch is almosttotally made up of amylopectin. Meanwhile, potato starch generallycomprises about 20% amylose, rice starch comprises anamylose:amylopectin ratio of about 20:80, and waxy rice starch comprisesonly about 2% amylose. Further, tapioca starch generally comprises about15% to about 18% amylose, and wheat starch has an amylose content ofaround 25%.

In some embodiments, the nutritional composition comprises gelatinizedand/or pre-gelatinized waxy corn starch. In other embodiments, thenutritional composition comprises gelatinized and/or pre-gelatinizedtapioca starch. Other gelatinized or pre-gelatinized starches, such asrice starch or potato starch may also be used.

Additionally, the nutritional compositions of the present disclosurecomprise at least one source of pectin. The source of pectin maycomprise any variety or grade of pectin known in the art. In someembodiments, the pectin has a degree of esterification of less than 50%and is classified as low methylated (“LM”) pectin. In some embodiments,the pectin has a degree of esterification of greater than or equal to50% and is classified as high-ester or high methylated (“HM”) pectin. Instill other embodiments, the pectin is very low (“VL”) pectin, which hasa degree of esterification that is less than approximately 15%. Further,the nutritional composition of the present disclosure may comprise LMpectin, HM pectin, VL pectin, or any mixture thereof. The nutritionalcomposition may include pectin that is soluble in water. And, as knownin the art, the solubility and viscosity of a pectin solution arerelated to the molecular weight, degree of esterification, concentrationof the pectin preparation and the pH and presence of counterions.

Moreover, pectin has a unique ability to form gels. Generally, undersimilar conditions, a pectin's degree of gelation, the gellingtemperature, and the gel strength are proportional to one another, andeach is generally proportional to the molecular weight of the pectin andinversely proportional to the degree of esterification. For example, asthe pH of a pectin solution is lowered, ionization of the carboxylategroups is repressed, and, as a result of losing their charge, saccharidemolecules do not repel each other over their entire length. Accordingly,the polysaccharide molecules can associate over a portion of theirlength to form a gel. Yet pectins with increasing degrees of methylationwill gel at somewhat higher pH because they have fewer carboxylateanions at any given pH. (J. N. Bemiller, An Introduction to Pectins:Structure and Properties, Chemistry and Function of Pectins; Chapter 1;1986.)

The nutritional composition may comprise a gelatinized and/orpregelatinized starch together with pectin and/or gelatinized pectin.While not wishing to be bound by this or any other theory, it isbelieved that the use of pectin, such as LM pectin, which is ahydrocolloid of large molecular weight, together with starch granules,provides a synergistic effect that increases the molecular internalfriction within a fluid matrix. The carboxylic groups of the pectin mayalso interact with calcium ions present in the nutritional composition,thus leading to an increase in viscosity, as the carboxylic groups ofthe pectin form a weak gel structure with the calcium ion(s), and alsowith peptides present in the nutritional composition. In someembodiments, the nutritional composition comprises a ratio of starch topectin that is between about 12:1 and 20:1, respectively. In otherembodiments, the ratio of starch to pectin is about 17:1. In someembodiments, the nutritional composition may comprise between about 0.05and about 2.0% w/w pectin. In a particular embodiment, the nutritionalcomposition may comprise about 0.5% w/w pectin.

Pectins for use herein typically have a peak molecular weight of 8,000Daltons or greater. The pectins of the present disclosure have apreferred peak molecular weight of between 8,000 and about 500,000, morepreferred is between about 10,000 and about 200,000 and most preferredis between about 15,000 and about 100,000 Daltons. In some embodiments,the pectin of the present disclosure may be hydrolyzed pectin. Incertain embodiments, the nutritional composition comprises hydrolyzedpectin having a molecular weight less than that of intact or unmodifiedpectin. The hydrolyzed pectin of the present disclosure can be preparedby any means known in the art to reduce molecular weight. Examples ofsaid means are chemical hydrolysis, enzymatic hydrolysis and mechanicalshear. A preferred means of reducing the molecular weight is by alkalineor neutral hydrolysis at elevated temperature. In some embodiments, thenutritional composition comprises partially hydrolyzed pectin. Incertain embodiments, the partially hydrolyzed pectin has a molecularweight that is less than that of intact or unmodified pectin but morethan 3,300 Daltons.

The nutritional composition may contain at least one acidicpolysaccharide. An acidic polysaccharide, such as negatively chargedpectin, may induce an anti-adhesive effect on pathogens in a subject'sgastrointestinal tract. Indeed, nonhuman milk acidic oligosaccharidesderived from pectin are able to interact with the epithelial surface andare known to inhibit the adhesion of pathogens on the epithelialsurface.

In some embodiments, the nutritional composition comprises at least onepectin-derived acidic oligosaccharide. Pectin-derived acidicoligosaccharide(s) (pAOS) result from enzymatic pectinolysis, and thesize of a pAOS depends on the enzyme use and on the duration of thereaction. In such embodiments, the pAOS may beneficially affect asubject's stool viscosity, stool frequency, stool pH and/or feedingtolerance. The nutritional composition of the present disclosure maycomprise between about 2 g pAOS per liter of formula and about 6 g pAOSper liter of formula. In an embodiment, the nutritional compositioncomprises about 0.2 g pAOS/dL, corresponding to the concentration ofacidic oligosaccharides in human milk. (Fanaro et al., “AcidicOligosaccharides from Pectin Hydrolysate as New Component for InfantFormulae: Effect on Intestinal Flora, Stool Characteristics, and pH”,Journal of Pediatric Gastroenterology and Nutrition, 41: 186-190, August2005)

In some embodiments, the nutritional composition comprises up to about20% w/w of a mixture of starch and pectin. In some embodiments, thenutritional composition comprises up to about 19% starch and up to about1% pectin. In other embodiments, the nutritional composition comprisesabout up to about 15% starch and up to about 5% pectin. In still otherembodiments, the nutritional composition comprises up to about 18%starch and up to about 2% pectin. In some embodiments the nutritionalcomposition comprises between about 0.05% w/w and about 20% w/w of amixture of starch and pectin. Other embodiments include between about0.05% and about 19% w/w starch and between about 0.05% and about 1% w/wpectin. Further, the nutritional composition may comprise between about0.05% and about 15% w/w starch and between about 0.05% and about 5% w/wpectin.

The disclosed nutritional composition(s) may be provided in any formknown in the art, such as a powder, a gel, a suspension, a paste, asolid, a liquid, a liquid concentrate, a reconstituteable powdered milksubstitute or a ready-to-use product. The nutritional composition may,in certain embodiments, comprise a nutritional supplement, children'snutritional product, infant formula, human milk fortifier, growing-upmilk or any other nutritional composition designed for an infant or apediatric subject. Nutritional compositions of the present disclosureinclude, for example, orally-ingestible, health-promoting substancesincluding, for example, foods, beverages, tablets, capsules and powders.Moreover, the nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form. Insome embodiments, the nutritional composition is in powder form with aparticle size in the range of 5 μm to 1500 μm, more preferably in therange of 10 μm to 300 μm.

If the nutritional composition is in the form of a ready-to-use product,the osmolality of the nutritional composition may be between about 100and about 1100 mOsm/kg water, more typically about 200 to about 700mOsm/kg water.

Suitable fat or lipid sources for the nutritional composition of thepresent disclosure may be any known or used in the art, including butnot limited to, animal sources, e.g., milk fat, butter, butter fat, eggyolk lipid; marine sources, such as fish oils, marine oils, single celloils; vegetable and plant oils, such as corn oil, canola oil, sunfloweroil, soybean oil, palm olein oil, coconut oil, high oleic sunflower oil,evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil,cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil,wheat germ oil; medium chain triglyceride oils and emulsions and estersof fatty acids; and any combinations thereof.

In some embodiments, the nutritional composition comprises at least oneadditional carbohydrate source, that is, a carbohydrate componentprovided in addition to the aforementioned starch component. Additionalcarbohydrate sources can be any used in the art, e.g., lactose, glucose,fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrupsolids, and the like. The amount of the additional carbohydratecomponent in the nutritional composition typically can vary from betweenabout 5 g and about 25 g/100 kcal. In some embodiments, the amount ofcarbohydrate is between about 6 g and about 22 g/100 kcal. In otherembodiments, the amount of carbohydrate is between about 12 g and about14 g/100 kcal. In some embodiments, corn syrup solids are preferred.Moreover, hydrolyzed, partially hydrolyzed, and/or extensivelyhydrolyzed carbohydrates may be desirable for inclusion in thenutritional composition due to their easy digestibility. Specifically,hydrolyzed carbohydrates are less likely to contain allergenic epitopes.

Non-limiting examples of carbohydrate materials suitable for use hereininclude hydrolyzed or intact, naturally or chemically modified, starchessourced from corn, tapioca, rice or potato, in waxy or non-waxy forms.Non-limiting examples of suitable carbohydrates include varioushydrolyzed starches characterized as hydrolyzed cornstarch,maltodextrin, maltose, corn syrup, dextrose, corn syrup solids, glucose,and various other glucose polymers and combinations thereof.Non-limiting examples of other suitable carbohydrates include thoseoften referred to as sucrose, lactose, fructose, high fructose cornsyrup, indigestible oligosaccharides such as fructooligosaccharides andcombinations thereof.

In one particular embodiment, the additional carbohydrate component ofthe nutritional composition is comprised of 100% lactose. In anotherembodiment, the additional carbohydrate component comprises betweenabout 0% and 60% lactose. In another embodiment, the additionalcarbohydrate component comprises between about 15% and 55% lactose. Inyet another embodiment, the additional carbohydrate component comprisesbetween about 20% and 30% lactose. In these embodiments, the remainingsource of carbohydrates may be any carbohydrate known in the art. In anembodiment, the carbohydrate component comprises about 25% lactose andabout 75% corn syrup solids.

In one embodiment, the nutritional composition may contain one or moreprobiotics. Any probiotic known in the art may be acceptable in thisembodiment. In a particular embodiment, the probiotic may be selectedfrom any Lactobacillus species, Lactobacillus rhamnosus GG (ATCC number53103), Bifidobacterium species, Bifidobacterium longum BB536 (BL999,ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382),Bifidobacterium breve AH1205 (NCIMB: 41387), Bifidobacterium infantis35624 (NCIMB: 41003), and Bifidobacterium animalis subsp. lactis BB-12(DSM No. 10140) or any combination thereof.

If included in the composition, the amount of the probiotic may varyfrom about 1×10⁴ to about 1×10¹⁰ colony forming units (cfu) per kg bodyweight per day. In another embodiment, the amount of the probiotic mayvary from about 10⁶ to about 10¹⁰ cfu per kg body weight per day. Instill another embodiment, the amount of the probiotic may vary fromabout 10⁷ to about 10⁹ cfu per day. In yet another embodiment, theamount of the probiotic may be at least about 10⁶ cfu per day. Incertain embodiments, the nutritional composition comprises between about1×10⁴ to about 1.5×10¹⁰ cfu of Lactobacillus rhamnosus GG per 100 kcal,more preferably from about 1×10⁶ to about 1×10⁹ cfu of Lactobacillusrhamnosus GG per 100 kcal.

In an embodiment, the probiotic(s) may be viable or non-viable. As usedherein, the term “viable”, refers to live microorganisms. The term“non-viable” or “non-viable probiotic” means non-living probioticmicroorganisms, their cellular components and/or metabolites thereof.Such non-viable probiotics may have been heat-killed or otherwiseinactivated, but they retain the ability to favorably influence thehealth of the host. The probiotics useful in the present disclosure maybe naturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such new source is now known or laterdeveloped.

The nutritional composition of the disclosure may contain a source oflong chain polyunsaturated fatty acid (LCPUFA) that comprisesdocosahexaenoic acid. Other suitable LCPUFAs include, but are notlimited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenicacid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA).

In an embodiment, especially if the nutritional composition is an infantformula, the nutritional composition is supplemented with both DHA andARA. In this embodiment, the weight ratio of ARA:DHA may be betweenabout 1:3 and about 9:1. In a particular embodiment, the ratio ofARA:DHA is from about 1:2 to about 4:1.

The amount of long chain polyunsaturated fatty acid in the nutritionalcomposition is advantageously at least about 5 mg/100 kcal, and may varyfrom about 5 mg/100 kcal to about 100 mg/100 kcal, more preferably fromabout 10 mg/100 kcal to about 50 mg/100 kcal.

The nutritional composition may be supplemented with oils containing DHAand/or ARA using standard techniques known in the art. For example, DHAand ARA may be added to the composition by replacing an equivalentamount of an oil, such as high oleic sunflower oil, normally present inthe composition. As another example, the oils containing DHA and ARA maybe added to the composition by replacing an equivalent amount of therest of the overall fat blend normally present in the compositionwithout DHA and ARA.

If utilized, the source of DHA and/or ARA may be any source known in theart such as marine oil, fish oil, single cell oil, egg yolk lipid, andbrain lipid. In some embodiments, the DHA and ARA are sourced fromsingle cell Martek oils, DHAS CO® and ARAS CO®, or variations thereof.The DHA and ARA can be in natural form, provided that the remainder ofthe LCPUFA source does not result in any substantial deleterious effecton the infant. Alternatively, the DHA and ARA can be used in refinedform.

In an embodiment, sources of DHA and ARA are single cell oils as taughtin U.S. Pat. Nos. 5,374,567; 5,550,156; and 5,397,591, the disclosuresof which are incorporated herein in their entirety by reference.However, the present disclosure is not limited to only such oils.

Furthermore, some embodiments of the nutritional composition may mimiccertain characteristics of human breast milk. However, to fulfill thespecific nutrient requirements of some subjects, the nutritionalcomposition may comprise a higher amount of some nutritional componentsthan does human milk. For example, the nutritional composition maycomprise a greater amount of DHA than does human breast milk.Accordingly, the enhanced level of DHA of the nutritional compositionmay compensate for an existing nutritional DHA deficit.

As noted, the disclosed nutritional composition may comprise a source ofβ-glucan. Glucans are polysaccharides, specifically polymers of glucose,which are naturally occurring and may be found in cell walls ofbacteria, yeast, fungi, and plants. Beta glucans (β-glucans) arethemselves a diverse subset of glucose polymers, which are made up ofchains of glucose monomers linked together via beta-type glycosidicbonds to form complex carbohydrates.

β-1,3-glucans are carbohydrate polymers purified from, for example,yeast, mushroom, bacteria, algae, or cereals. (Stone B A, Clarke A E.Chemistry and Biology of (1-3)-Beta-Glucans. London:Portland Press Ltd;1993.) The chemical structure of β-1,3-glucan depends on the source ofthe β-1,3-glucan. Moreover, various physiochemical parameters, such assolubility, primary structure, molecular weight, and branching, play arole in biological activities of β-1,3-glucans. (Yadomae T., Structureand biological activities of fungal beta-1,3-glucans. Yakugaku Zasshi.2000; 120:413-431.)

β-1,3-glucans are naturally occurring polysaccharides, with or withoutβ-1,6-glucose side chains that are found in the cell walls of a varietyof plants, yeasts, fungi and bacteria. β-1,3;1,6-glucans are thosecontaining glucose units with (1,3) links having side chains attached atthe (1,6) position(s). β-1,3;1,6 glucans are a heterogeneous group ofglucose polymers that share structural commonalities, including abackbone of straight chain glucose units linked by a β-1,3 bond withβ-1,6-linked glucose branches extending from this backbone. While thisis the basic structure for the presently described class of β-glucans,some variations may exist. For example, certain yeast β-glucans haveadditional regions of β(1,3) branching extending from the β(1,6)branches, which add further complexity to their respective structures.

β-glucans derived from baker's yeast, Saccharomyces cerevisiae, are madeup of chains of D-glucose molecules connected at the 1 and 3 positions,having side chains of glucose attached at the 1 and 6 positions.Yeast-derived β-glucan is an insoluble, fiber-like, complex sugar havingthe general structure of a linear chain of glucose units with a β-1,3backbone interspersed with β-1,6 side chains that are generally 6-8glucose units in length. More specifically, β-glucan derived frombaker's yeast is poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D-glucopyranose.

Furthermore, β-glucans are well tolerated and do not produce or causeexcess gas, abdominal distension, bloating or diarrhea in pediatricsubjects. Addition of β-glucan to a nutritional composition for apediatric subject, such as an infant formula, a growing-up milk oranother children's nutritional product, will improve the subject'simmune response by increasing resistance against invading pathogens andtherefore maintaining or improving overall health.

The nutritional composition of the present disclosure comprisesβ-glucan. In some embodiments, the β-glucan is β-1,3;1,6-glucan. In someembodiments, the β-1,3;1,6-glucan is derived from baker's yeast. Thenutritional composition may comprise whole glucan particle β-glucan,particulate β-glucan, PGG-glucan(poly-1,6-β-D-glucopyranosyl-1,3-β-D-glucopyranose) or any mixturethereof.

In some embodiments, the amount of β-glucan present in the compositionis at between about 0.010 and about 0.080 g per 100 g of composition. Inother embodiments, the nutritional composition comprises between about10 and about 30 mg β-glucan per serving. In another embodiment, thenutritional composition comprises between about 5 and about 30 mgβ-glucan per 8 fl. oz. (236.6 mL) serving. In other embodiments, thenutritional composition comprises an amount of β-glucan sufficient toprovide between about 15 mg and about 90 mg O-glucan per day. Thenutritional composition may be delivered in multiple doses to reach atarget amount of β-glucan delivered to the subject throughout the day.

In some embodiments, the amount of β-glucan in the nutritionalcomposition is between about 3 mg and about 17 mg per 100 kcal. Inanother embodiment the amount of β-glucan is between about 6 mg andabout 17 mg per 100 kcal.

One or more vitamins and/or minerals may also be added in to thenutritional composition in amounts sufficient to supply the dailynutritional requirements of a subject. It is to be understood by one ofordinary skill in the art that vitamin and mineral requirements willvary, for example, based on the age of the child. For instance, aninfant may have different vitamin and mineral requirements than a childbetween the ages of one and thirteen years. Thus, the embodiments arenot intended to limit the nutritional composition to a particular agegroup but, rather, to provide a range of acceptable vitamin and mineralcomponents.

The nutritional composition may optionally include, but is not limitedto, one or more of the following vitamins or derivations thereof:vitamin B₁ (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate,TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B₂(riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide,FAD, lactoflavin, ovoflavin), vitamin B₃ (niacin, nicotinic acid,nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD,nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid),vitamin B₃-precursor tryptophan, vitamin B₆ (pyridoxine, pyridoxal,pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate,panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitaminB₁₂ (cobalamin, methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid),vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esterswith other long-chain fatty acids, retinal, retinoic acid, retinolesters), vitamin D (calciferol, cholecalciferol, vitamin D₃,1,25,-dihydroxyvitamin D), vitamin E (α-tocopherol, α-tocopherolacetate, α-tocopherol succinate, α-tocopherol nicotinate, α-tocopherol),vitamin K (vitamin K₁, phylloquinone, naphthoquinone, vitamin K₂,menaquinone-7, vitamin K₃, menaquinone-4, menadione, menaquinone-8,menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,6-carotene and any combinations thereof.

Further, the nutritional composition may optionally include, but is notlimited to, one or more of the following minerals or derivationsthereof: boron, calcium, calcium acetate, calcium gluconate, calciumchloride, calcium lactate, calcium phosphate, calcium sulfate, chloride,chromium, chromium chloride, chromium picolonate, copper, coppersulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyliron, ferric iron, ferrous fumarate, ferric orthophosphate, irontrituration, polysaccharide iron, iodide, iodine, magnesium, magnesiumcarbonate, magnesium hydroxide, magnesium oxide, magnesium stearate,magnesium sulfate, manganese, molybdenum, phosphorus, potassium,potassium phosphate, potassium iodide, potassium chloride, potassiumacetate, selenium, sulfur, sodium, docusate sodium, sodium chloride,sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate andmixtures thereof. Non-limiting exemplary derivatives of mineralcompounds include salts, alkaline salts, esters and chelates of anymineral compound.

The minerals can be added to nutritional compositions in the form ofsalts such as calcium phosphate, calcium glycerol phosphate, sodiumcitrate, potassium chloride, potassium phosphate, magnesium phosphate,ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, andsodium selenite. Additional vitamins and minerals can be added as knownwithin the art.

In an embodiment, the nutritional composition may contain between about10 and about 50% of the maximum dietary recommendation for any givencountry, or between about 10 and about 50% of the average dietaryrecommendation for a group of countries, per serving of vitamins A, C,and E, zinc, iron, iodine, selenium, and choline. In another embodiment,the children's nutritional composition may supply about 10-30% of themaximum dietary recommendation for any given country, or about 10-30% ofthe average dietary recommendation for a group of countries, per servingof B-vitamins. In yet another embodiment, the levels of vitamin D,calcium, magnesium, phosphorus, and potassium in the children'snutritional product may correspond with the average levels found inmilk. In other embodiments, other nutrients in the children'snutritional composition may be present at about 20% of the maximumdietary recommendation for any given country, or about 20% of theaverage dietary recommendation for a group of countries, per serving.

The nutritional compositions of the present disclosure may optionallyinclude one or more of the following flavoring agents, including, butnot limited to, flavored extracts, volatile oils, cocoa or chocolateflavorings, peanut butter flavoring, cookie crumbs, vanilla or anycommercially available flavoring. Examples of useful flavorings include,but are not limited to, pure anise extract, imitation banana extract,imitation cherry extract, chocolate extract, pure lemon extract, pureorange extract, pure peppermint extract, honey, imitation pineappleextract, imitation rum extract, imitation strawberry extract, or vanillaextract; or volatile oils, such as balm oil, bay oil, bergamot oil,cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil;peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch,toffee, and mixtures thereof. The amounts of flavoring agent can varygreatly depending upon the flavoring agent used. The type and amount offlavoring agent can be selected as is known in the art.

The nutritional compositions of the present disclosure may optionallyinclude one or more emulsifiers that may be added for stability of thefinal product. Examples of suitable emulsifiers include, but are notlimited to, lecithin (e.g., from egg or soy), alpha lactalbumin and/ormono- and di-glycerides, and mixtures thereof. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

The nutritional compositions of the present disclosure may optionallyinclude one or more preservatives that may also be added to extendproduct shelf life. Suitable preservatives include, but are not limitedto, potassium sorbate, sodium sorbate, potassium benzoate, sodiumbenzoate, calcium disodium EDTA, and mixtures thereof.

The nutritional compositions of the present disclosure may optionallyinclude one or more stabilizers. Suitable stabilizers for use inpracticing the nutritional composition of the present disclosureinclude, but are not limited to, gum arabic, gum ghatti, gum karaya, gumtragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum,pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC(sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid estersof mono- and diglycerides), dextran, carrageenans, and mixtures thereof.

The nutritional compositions of the disclosure may provide minimal,partial or total nutritional support. The compositions may benutritional supplements or meal replacements. The compositions may, butneed not, be nutritionally complete. In an embodiment, the nutritionalcomposition of the disclosure is nutritionally complete and containssuitable types and amounts of lipid, carbohydrate, protein, vitamins andminerals. The amount of lipid or fat typically can vary from about 1 toabout 7 g/100 kcal. The amount of protein typically can vary from about1 to about 7 g/100 kcal. The amount of carbohydrate typically can varyfrom about 6 to about 22 g/100 kcal.

The nutritional composition of the present disclosure may furtherinclude at least one additional phytonutrient, that is, anotherphytonutrient component in addition to the pectin and/or starchcomponents described hereinabove. Phytonutrients, or their derivatives,conjugated forms or precursors, that are identified in human milk arepreferred for inclusion in the nutritional composition. Typically,dietary sources of carotenoids and polyphenols are absorbed by a nursingmother and retained in milk, making them available to nursing infants.Addition of these phytonutrients to infant or children's formulas allowssuch formulas to mirror the composition and functionality of human milkand to promote general health and well being.

For example, in some embodiments, the nutritional composition of thepresent disclosure may comprise, in an 8 fl. oz. (236.6 mL) serving,between about 80 and about 300 mg anthocyanins, between about 100 andabout 600 mg proanthocyanidins, between about 50 and about 500 mgflavan-3-ols, or any combination or mixture thereof. In otherembodiments, the nutritional composition comprises apple extract, grapeseed extract, or a combination or mixture thereof. Further, the at leastone phytonutrient of the nutritional composition may be derived from anysingle or blend of fruit, grape seed and/or apple or tea extract(s).

For the purposes of this disclosure, additional phytonutrients may beadded to a nutritional composition in native, purified, encapsulatedand/or chemically or enzymatically-modified form so as to deliver thedesired sensory and stability properties. In the case of encapsulation,it is desirable that the encapsulated phytonutrients resist dissolutionwith water but are released upon reaching the small intestine. Thiscould be achieved by the application of enteric coatings, such ascross-linked alginate and others.

Examples of additional phytonutrients suitable for the nutritionalcomposition include, but are not limited to, anthocyanins,proanthocyanidins, flavan-3-ols (i.e. catechins, epicatechins, etc.),flavanones, flavonoids, isoflavonoids, stilbenoids (i.e. resveratrol,etc.) proanthocyanidins, anthocyanins, resveratrol, quercetin, curcumin,and/or any mixture thereof, as well as any possible combination ofphytonutrients in a purified or natural form. Certain components,especially plant-based components of the nutritional compositions mayprovide a source of phytonutrients.

Some amounts of phytonutrients may be inherently present in knowningredients, such as natural oils, that are commonly used to makenutritional compositions for pediatric subjects. These inherentphytonutrient(s) may be but are not necessarily considered part of thephytonutrient component described in the present disclosure. In someembodiments, the phytonutrient concentrations and ratios as describedherein are calculated based upon added and inherent phytonutrientsources. In other embodiments, the phytonutrient concentrations andratios as described herein are calculated based only upon addedphytonutrient sources.

In some embodiments, the nutritional composition comprises anthocyanins,such as, for example, glucosides of aurantinidin, cyanidin, delphinidin,europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin,and rosinidin. These and other anthocyanins suitable for use in thenutritional composition are found in a variety of plant sources.Anthocyanins may be derived from a single plant source or a combinationof plant sources. Non-limiting examples of plants rich in anthocyaninssuitable for use in the inventive composition include: berries (acai,grape, bilberry, blueberry, lingonberry, black currant, chokeberry,blackberry, raspberry, cherry, red currant, cranberry, crowberry,cloudberry, whortleberry, rowanberry), purple corn, purple potato,purple carrot, red sweet potato, red cabbage, eggplant.

In some embodiments, the nutritional composition of the presentdisclosure comprises proanthocyanidins, which include but are notlimited to flavan-3-ols and polymers of flavan-3-ols (e.g., catechins,epicatechins) with degrees of polymerization in the range of 2 to 11.Such compounds may be derived from a single plant source or acombination of plant sources. Non-limiting examples of plant sourcesrich in proanthocyanidins suitable for use in the inventive nutritionalcomposition include: grape, grape skin, grape seed, green tea, blacktea, apple, pine bark, cinnamon, cocoa, bilberry, cranberry, blackcurrant chokeberry.

Non-limiting examples of flavan-3-ols which are suitable for use in theinventive nutritional composition include catechin, epicatechin,gallocatechin, epigallocatechin, epicatechin gallate,epicatechin-3-gallate, epigallocatechin and gallate. Plants rich in thesuitable flavan-3-ols include, but are not limited to, teas, red grapes,cocoa, green tea, apricot and apple.

Certain polyphenol compounds, in particular flavan-3-ols, may improvelearning and memory in a human subject by increasing brain blood flow,which is associated with an increase and sustained brain energy/nutrientdelivery as well as formation of new neurons. Polyphenols may alsoprovide neuroprotective actions and may increase both brainsynaptogenesis and antioxidant capability, thereby supporting optimalbrain development in younger children.

Preferred sources of flavan-3-ols for the nutritional compositioninclude at least one apple extract, at least one grape seed extract or amixture thereof. For apple extracts, flavan-3-ols are broken down intomonomers occurring in the range 4% to 20% and polymers in the range 80%to 96%. For grape seed extracts flavan-3-ols are broken down intomonomers (about 46%) and polymers (about 54%) of the total favan-3-olsand total polyphenolic content. Preferred degree of polymerization ofpolymeric flavan-3-ols is in the range of between about 2 and 11.Furthermore, apple and grape seed extracts may contain catechin,epicatechin, epigallocatechin, epicatechin gallate, epigallocatechingallate, polymeric proanthocyanidins, stilbenoids (i.e. resveratrol),flavonols (i.e. quercetin, myricetin), or any mixture thereof. Plantsources rich in flavan-3-ols include, but are not limited to apple,grape seed, grape, grape skin, tea (green or black), pine bark,cinnamon, cocoa, bilberry, cranberry, black currant, chokeberry.

If the nutritional composition is administered to a pediatric subject,an amount of flavan-3-ols, including monomeric flavan-3-ols, polymericflavan-3-ols or a combination thereof, ranging from between about 0.01mg and about 450 mg per day may be administered. In some cases, theamount of flavan-3-ols administered to an infant or child may range fromabout 0.01 mg to about 170 mg per day, from about 50 to about 450 mg perday, or from about 100 mg to about 300 mg per day.

In an embodiment of the disclosure, flavan-3-ols are present in thenutritional composition in an amount ranging from about 0.4 to about 3.8mg/g nutritional composition (about 9 to about 90 mg/100 kcal). Inanother embodiment, flavan-3-ols are present in an amount ranging fromabout 0.8 to about 2.5 mg/g nutritional composition (about 20 to about60 mg/100 kcal).

In some embodiments, the nutritional composition of the presentdisclosure comprises flavanones. Non-limiting examples of suitableflavanones include butin, eriodictyol, hesperetin, hesperidin,homeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin,poncirin, sakuranetin, sakuranin, steurbin. Plant sources rich inflavanones include, but are not limited to orange, tangerine,grapefruit, lemon, lime. The nutritional composition may be formulatedto deliver between about 0.01 and about 150 mg flavanones per day.

Moreover, the nutritional composition may also comprise flavonols.Flavonols from plant or algae extracts may be used. Flavonols, such asishrhametin, kaempferol, myricetin, quercetin, may be included in thenutritional composition in amounts sufficient to deliver between about0.01 and 150 mg per day to a subject.

The phytonutrient component of the nutritional composition may alsocomprise phytonutrients that have been identified in human milk,including but not limited to naringenin, hesperetin, anthocyanins,quercetin, kaempferol, epicatechin, epigallocatechin,epicatechin-gallate, epigallocatechin-gallate or any combinationthereof. In certain embodiments, the nutritional composition comprisesbetween about 50 and about 2000 nmol/L epicatechin, between about 40 andabout 2000 nmol/L epicatechin gallate, between about 100 and about 4000nmol/L epigallocatechin gallate, between about 50 and about 2000 nmol/Lnaringenin, between about 5 and about 500 nmol/L kaempferol, betweenabout 40 and about 4000 nmol/L hesperetin, between about 25 and about2000 nmol/L anthocyanins, between about 25 and about 500 nmol/Lquercetin, or a mixture thereof. Furthermore, the nutritionalcomposition may comprise the metabolite(s) of a phytonutrient or of itsparent compound, or it may comprise other classes of dietaryphytonutrients, such as glucosinolate or sulforaphane.

In certain embodiments, the nutritional composition comprisescarotenoids, such as lutein, zeaxanthin, astaxanthin, lycopene,beta-carotene, alpha-carotene, gamma-carotene, and/orbeta-cryptoxanthin. Plant sources rich in carotenoids include, but arenot limited to kiwi, grapes, citrus, tomatoes, watermelons, papayas andother red fruits, or dark greens, such as kale, spinach, turnip greens,collard greens, romaine lettuce, broccoli, zucchini, garden peas andBrussels sprouts, spinach, carrots.

Humans cannot synthesize carotenoids, but over 34 carotenoids have beenidentified in human breast milk, including isomers and metabolites ofcertain carotenoids. In addition to their presence in breast milk,dietary carotenoids, such as alpha and beta-carotene, lycopene, lutein,zeaxanthin, astaxanthin, and cryptoxanthin are present in serum oflactating women and breastfed infants. Carotenoids in general have beenreported to improve cell-to-cell communication, promote immune function,support healthy respiratory health, protect skin from UV light damage,and have been linked to reduced risk of certain types of cancer, andall-cause mortality. Furthermore, dietary sources of carotenoids and/orpolyphenols are absorbed by human subjects, accumulated and retained inbreast milk, making them available to nursing infants. Thus, addition ofphytonutrients to infant formulas or children's products would bring theformulas closer in composition and functionality to human milk.

Flavonoids, as a whole, may also be included in the nutritionalcomposition, as flavonoids cannot be synthesized by humans. Moreover,flavonoids from plant or algae extracts may be useful in the monomer,dimer and/or polymer forms. In some embodiments, the nutritionalcomposition comprises levels of the monomeric forms of flavonoidssimilar to those in human milk during the first three months oflactation. Although flavonoid aglycones (monomers) have been identifiedin human milk samples, the conjugated forms of flavonoids and/or theirmetabolites may also be useful in the nutritional composition. Theflavonoids could be added in the following forms: free, glucuronides,methyl glucuronides, sulphates, and methyl sulphates.

The nutritional composition may also comprise isoflavonoids and/orisoflavones. Examples include, but are not limited to, genistein(genistin), daidzein (daidzin), glycitein, biochanin A, formononetin,coumestrol, irilone, orobol, pseudobaptigenin, anagyroidisoflavone A andB, calycosin, glycitein, irigenin, 5-O-methylgenistein, pratensein,prunetin, psi-tectorigenin, retusin, tectorigenin, iridin, ononin,puerarin, tectoridin, derrubone, luteone, wighteone, alpinumisoflavone,barbigerone, di-O-methylalpinumisoflavone, and4′-methyl-alpinumisoflavone. Plant sources rich in isoflavonoids,include, but are not limited to, soybeans, psoralea, kudzu, lupine,fava, chick pea, alfalfa, legumes and peanuts. The nutritionalcomposition may be formulated to deliver between about 0.01 and about150 mg isoflavones and/or isoflavonoids per day.

In an embodiment, the nutritional composition(s) of the presentdisclosure comprises an effective amount of choline. Choline is anutrient that is essential for normal function of cells. It is aprecursor for membrane phospholipids, and it accelerates the synthesisand release of acetylcholine, a neurotransmitter involved in memorystorage. Moreover, though not wishing to be bound by this or any othertheory, it is believed that dietary choline and docosahexaenoic acid(DHA) act synergistically to promote the biosynthesis ofphosphatidylcholine and thus help promote synaptogenesis in humansubjects. Additionally, choline and DHA may exhibit the synergisticeffect of promoting dendritic spine formation, which is important in themaintenance of established synaptic connections. In some embodiments,the nutritional composition(s) of the present disclosure includes aneffective amount of choline, which is about 20 mg choline per 8 fl. oz.(236.6 mL) serving to about 100 mg per 8 fl. oz. (236.6 mL) serving.

Moreover, in some embodiments, the nutritional composition isnutritionally complete, containing suitable types and amounts of lipids,carbohydrates, proteins, vitamins and minerals to be a subject's solesource of nutrition. Indeed, the nutritional composition may optionallyinclude any number of proteins, peptides, amino acids, fatty acids,probiotics and/or their metabolic by-products, prebiotics, carbohydratesand any other nutrient or other compound that may provide manynutritional and physiological benefits to a subject. Further, thenutritional composition of the present disclosure may comprise flavors,flavor enhancers, sweeteners, pigments, vitamins, minerals, therapeuticingredients, functional food ingredients, food ingredients, processingingredients or combinations thereof.

The present disclosure further provides a method for providingnutritional support to a subject. The method includes administering tothe subject an effective amount of the nutritional composition of thepresent disclosure.

The nutritional composition may be expelled directly into a subject'sintestinal tract. In some embodiments, the nutritional composition isexpelled directly into the gut. In some embodiments, the composition maybe formulated to be consumed or administered enterally under thesupervision of a physician and may be intended for the specific dietarymanagement of a disease or condition, such as celiac disease and/or foodallergy, for which distinctive nutritional requirements, based onrecognized scientific principles, are established by medical evaluation.

The nutritional composition of the present disclosure is not limited tocompositions comprising nutrients specifically listed herein. Anynutrients may be delivered as part of the composition for the purpose ofmeeting nutritional needs and/or in order to optimize the nutritionalstatus in a subject.

In some embodiments, the nutritional composition may be delivered to aninfant from birth until a time that matches full-term gestation. In someembodiments, the nutritional composition may be delivered to an infantuntil at least about three months corrected age. In another embodiment,the nutritional composition may be delivered to a subject as long as isnecessary to correct nutritional deficiencies. In yet anotherembodiment, the nutritional composition may be delivered to an infantfrom birth until at least about six months corrected age. In yet anotherembodiment, the nutritional composition may be delivered to an infantfrom birth until at least about one year corrected age.

The nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form.

In some embodiments, the nutritional composition of the presentdisclosure is a growing-up milk. Growing-up milks are fortifiedmilk-based beverages intended for children over 1 year of age (typicallyfrom 1-3 years of age, from 4-6 years of age or from 1-6 years of age).They are not medical foods and are not intended as a meal replacement ora supplement to address a particular nutritional deficiency. Instead,growing-up milks are designed with the intent to serve as a complementto a diverse diet to provide additional insurance that a child achievescontinual, daily intake of all essential vitamins and minerals,macronutrients plus additional functional dietary components, such asnon-essential nutrients that have purported health-promoting properties.

The exact composition of a nutritional composition according to thepresent disclosure can vary from market-to-market, depending on localregulations and dietary intake information of the population ofinterest. In some embodiments, nutritional compositions according to thedisclosure consist of a milk protein source, such as whole or skim milk,plus added sugar and sweeteners to achieve desired sensory properties,and added vitamins and minerals. The fat composition is typicallyderived from the milk raw materials. Total protein can be targeted tomatch that of human milk, cow milk or a lower value. Total carbohydrateis usually targeted to provide as little added sugar, such as sucrose orfructose, as possible to achieve an acceptable taste. Typically, VitaminA, calcium and Vitamin D are added at levels to match the nutrientcontribution of regional cow milk. Otherwise, in some embodiments,vitamins and minerals can be added at levels that provide approximately20% of the dietary reference intake (DRI) or 20% of the Daily Value (DV)per serving. Moreover, nutrient values can vary between marketsdepending on the identified nutritional needs of the intendedpopulation, raw material contributions and regional regulations.

In certain embodiments, the nutritional composition is hypoallergenic.In other embodiments, the nutritional composition is kosher. In stillfurther embodiments, the nutritional composition is a non-geneticallymodified product. In an embodiment, the nutritional formulation issucrose-free. The nutritional composition may also be lactose-free. Inother embodiments, the nutritional composition does not contain anymedium-chain triglyceride oil. In some embodiments, no carrageenan ispresent in the composition. In other embodiments, the nutritionalcomposition is free of all gums.

In some embodiments, the disclosure is directed to a staged nutritionalfeeding regimen for a pediatric subject, such as an infant or child,which includes a plurality of different nutritional compositionsaccording to the present disclosure. Each nutritional compositioncomprises a hydrolyzed protein, at least one pre-gelatinized starch, andat least one pectin. In certain embodiments, the nutritionalcompositions of the feeding regimen may also include a source of longchain polyunsaturated fatty acid, at least one prebiotic, an ironsource, a source of β-glucan, vitamins or minerals, lutein, zeaxanthin,or any other ingredient described hereinabove. The nutritionalcompositions described herein may be administered once per day or viaseveral administrations throughout the course of a day.

Examples are provided to illustrate some embodiments of the nutritionalcomposition of the present disclosure but should not be interpreted asany limitation thereon. Other embodiments within the scope of the claimsherein will be apparent to one skilled in the art from the considerationof the specification or practice of the nutritional composition ormethods disclosed herein. It is intended that the specification,together with the example, be considered to be exemplary only, with thescope and spirit of the disclosure being indicated by the claims whichfollow the example.

Example 1

This example illustrates an embodiment of a nutritional productaccording to the present disclosure.

Description kg per 100 kg carbohydrate, total 38.9 protein, total 28.8fat, total 25.6 prebiotics 4.5 soy lecithin 0.8 lactoferrin 0.3 calciumcarbonate 0.5 potassium citrate 0.2 ferrous sulfate 0.05 potassiumchloride 0.048 magnesium oxide 0.023 sodium chloride 0.025 zinc sulfate0.015 cupric sulfate 0.002 manganese sulfate 0.0003 sodium selenite0.00003 choline chloride 0.144 ascorbic acid 0.093 Niacinamide 0.006calcium pantothenate 0.003 vitamin A palmitate 0.007 vitamin B12 0.002vitamin D3 0.000001 Riboflavin 0.0008 thiamin 0.0006 vitamin B6 0.0004folic acid 0.0001 vitamin K1 0.006 biotin 0.00002 inositol 0.03 vitaminE acetate 0.01 taurine 0.05 L-carnitine 0.001

Example 2

This example illustrates another embodiment of a nutritional productaccording to the present disclosure.

Description kg per 100 kg carbohydrate, total 24.7 protein, total 31.9fat, total 39.3 prebiotics 3.6 lactoferrin 0.1 calcium carbonate 0.15ferrous sulfate 0.03 zinc sulfate 0.01 copper sulfate 0.00025 manganesesulfate 0.0002 sodium selenite 0.00001 choline bitartrate 0.05 ascorbicacid 0.004 sodium ascorbate 0.04 niacinamide 0.007 calcium pantothenate0.0005 vitamin A palmitate 0.0005 vitamin D3 0.0002 riboflavin 0.0001thiamin 0.00005 vitamin B6 0.00005 folic acid 0.000067 vitamin K10.00002 vitamin E acetate 0.008 taurine 0.02 fish oil 0.2 B-glucan 0.03

Example 3

This example illustrates one embodiment of ingredients that can be usedto prepare the nutritional product according to the present disclosure.

water 872 ml lactose 65.6 mg vegetable oil blend 353.0 mg  nonfat milkevaporated 34.0 mg whey protein concentrate  8.5 mggalacto-oligosaccharide  4.7 mg casein  3.5 mg polydextrose  2.4 mglactoferrin solution (10%)  1.0 mg single cell DHA and ARA oil blend0.94 mg mono- and di-glycerides  0.7 mg calcium carbonate 0.44 mgcalcium phosphate  0.4 mg potassium citrate  0.4 mg potassium chloride 0.4 mg soy lecithin  0.4 mg sodium chloride  0.3 mg potassium phosphate 0.3 mg choline chloride  0.2 mg magnesium oxide 0.08 mg calciumhydroxide 0.08 mg ferrous sulfate 0.07 mg

Example 4

This example illustrates another embodiment of ingredients that can beused to prepare the nutritional product according to the presentdisclosure.

water 686 ml reduced minerals whey  215 mg nonfat milk evaporated   67mg vegetable oil blend   33 mg lactose   17 mg galacto-oligosaccharide 4.7 mg polydextrose  2.4 mg lactoferrin solution (10%)  1.0 mg singlecell DHA and ARA oil blend  0.9 mg mono- and di-glycerides  0.7 mgcalcium carbonate 0.44 mg calcium phosphate  0.4 mg potassium citrate 0.4 mg potassium chloride  0.4 mg soy lecithin  0.4 mg potassiumphosphate  0.3 mg carrageenan  0.3 mg sodium citrate  0.2 mg cholinechloride  0.2 mg magnesium oxide 0.08 mg calcium chloride 0.08 mgferrous sulfate 0.07 mg

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Although embodiments of the disclosure have been described usingspecific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present disclosure, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedin whole or in part. For example, while methods for the production of acommercially sterile liquid nutritional supplement made according tothose methods have been exemplified, other uses are contemplated.Therefore, the spirit and scope of the appended claims should not belimited to the description of the versions contained therein.

1-11. (canceled)
 12. A method for modulating plasma corticosteronelevels in a pediatric subject, the method comprising administering tothe pediatric subject a milk-based nutritional composition comprisingbovine lactoferrin.
 13. The method according to claim 12, wherein thenutritional composition further comprises at least one prebiotic. 14.The method according to claim 13, wherein the at least one prebiotic ispresent at a level of about 0.1 g/100 kcal to about 1 g/100 kcal. 15.The method according to claim 13, wherein the at least one prebioticcomprises polydextrose.
 16. The method according to claim 15, whereinthe at least one prebiotic further comprises galactooligosaccharide. 17.The method according to claim 15, wherein polydextrose comprises atleast about 20% w/w of the at least one prebiotic.
 18. A method forsupporting gastrointestinal development in a pediatric subject, themethod comprising administering to the pediatric subject a milk-basednutritional composition comprising bovine lactoferrin and a prebioticcomponent comprising polydextrose.
 19. The method according to claim 18,wherein polydextrose comprises at least 20% w/w of the prebioticcomponent.
 20. The method according to claim 18, wherein the prebioticcomponent further comprises galactooligosaccharide.